Clinical Effects of Probiotics: A Pediatrician's Perspectivep

Kalliomäki Marko, MD, PhD, Adjunct professor of paediatrics, Consultant in paediatric gastroenterology, Department of Paediatrics, Functional Foods Forum, University of Turku and Turku University Hospital

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The contents of the presentation

• Role of gut microbiota in the development of gut and its immune system

• Potential effects of probiotics on gut immune Potential effects of probiotics on gut immune system

• Randomized clinical studies with probioticsdi h– diarrhea

– inflammatory bowel disease– irritable bowel syndrome– necrotizing enterocolitis (NEC)– allergic diseases

• Conclusions

Gut and its microbiota form immunologically active ecosystemGut and its microbiota form immunologically active ecosystem

• Adult human gut harbours 1-2 kilograms of bacteria in the gut

• There are 10 x more prokaryotic cells in the ut than eukary tic cells in the in the gut than eukaryotic cells in the whole body

• The number of bacterial genes in the gut (microbiome) outnumber that of g ( )human genes (genome) by a factor of 100 - humans have been called ’supraorganisms’

• GALT (gut associated lymphatic tissue) GALT (gut associated lymphatic tissue) forms a major part of the immune system (e.g. three fourths of the body’s plasma cells are located in the GALT)

Figure from Allergy 2007;62:1223-36:

BUT...

Development of mucosal immunity is immature at birth(The figure is a generous courtesy from prof. Allan Walker, Harvard Medical School;modified from Walker WA and Sanderson I, 2003)

Gut microbiota develops after birth

Breast-fed infants Formula-fed infants

Pediatrics 1983;72:317-321

NN

Development of the gut and its immune system is dependent on luminal microbial and dietary antigens (modified from Szepfalusi Z 2008)

C ll C ll Late InfancyLate Infancy

NeonateNeonate

Mucosal cell Mucosal cell

Cell enzyme Cell enzyme activity, mucus activity, mucus

productionproduction

Lamina propria Lamina propria ll l i ( ll l i (

Mucosal cell Mucosal cell turnoverturnover

cellularity (e.g. cellularity (e.g. regulatory T cells)regulatory T cells)

M l M l

Secretory IgASecretory IgA

Muscle Muscle wall, wall,

angiogeneangiogenesissis

Two functional arms of immune system in the gut

(Th fi f T ji NM d K k A 2008)(The figure from Tsuji NM and Kosaka A 2008)o Production ofsecretory IgAo Tolerance to luminal microbial and dietary antigens

- regulatory T cells are mandatory for 1) oral tolerance and 2) prevention of both h1 (f d d b ll ) d h2 k d ( ll )Th1- (found e.g. in diabetes mellitus) and Th2-skewed immune system (allergy)

- development and function of regulatory T cells is dependent on gut microbiota (Östman S et al. 2006, Ishikawa H et al. 2008)

Definition of probiotics

FAO/WHO (2001)Probiotic bacteria are live microorganisms which when Probiotic bacteria are live microorganisms which when administered in adequate amounts confer a health benefit on the host

Lactobacilli and bifidobacteria have been a part of Lactobacilli and bifidobacteria have been a part of human diet for millenia

• Historian Plinius (76 BC) recommended sour milk products for treatment of diarrhea

N b l l t Eli M t h ik ff • Nobel laureate Elie Metchnikoff (1908) claimed that yogurt promote health and longevity (autointoxication, Bulgarian bacillus)

• Frenchman Henry Tissier found bifidobacteria and showed their abundance in breastfed infants. He also recommended bifido-He also recommended bifidobacteria for treatment of diarrhea in infants (C R Soc Biol 1906) Figure from Wikipedia

Probiotics in the treatment and prevention of Probiotics in the treatment and prevention of diarrhea in children (Szajewska H et al. 2005, 2006)

• Probiotics in acute diarrhea in children:– have a moderate effect that depends on the strain used

(Lactobacillus GG, Lactobacillus reuteri, Saccharomyces boulardii) and dosedose.

– help against watery viral diarrhea, especially one caused by rota virus

– shorten the duration of illness approximately by one day if pp y y yadministered early in the course of disease

• Saccharomyces boulardii, Lactobacillus rhamnosus GG and Bifibobacterium lactis + Streptococcuc thermophilus prevent moderately antibiotic-associated diarrhea in children (NNT 7)moderately antibiotic associated diarrhea in children (NNT 7)

Potential mechanisms of action of probiotics in the gastrointestinal tract (Kalliomäki and Walker 2005)

1. Processing of enteral antigens2. Inhibition of attachment of other

bacteria to IEC3. Enhancement of IEC’s permeability 11113. Enhancement of IEC s permeability

barrier4. Suppression of pro-inflammatory

responses in IEC5. Modulation of IEC’s apoptosis

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5. Modulation of IEC s apoptosis6. Maturation of dendritic cells with

anti-allergic properties7. Production of regulatory cytokines

by immune cells in lamina propriaby immune cells in lamina propria

Proc Natl Acad Sci U S A. 2009 Feb 17;106(7):2371-2376. Epub 2009 Feb 3.

Dose ≈ 2 x 1012

6 h after the consumption

Induction of NF-κB pathways was dependent on p y pgrowth phase of the probiotic strain Lactobacillus

plantarum WCFS1

• Dead bacteria (heat-killed in the stationary phase of growth) induced almost all NF-κB subunits but also 3 antagonists of NF-κB

Proc Natl Acad Sci U S A. 2009 Feb 17;106(7):2371-6.

g f Factivity

• Bacteria in the stationary phase of the growth induced 2 NF-κB subunits and 3 antagonists of the NF-κB

ti itactivity• Bacteria in the logarithmic-phase of

growth (expotentially growing; midlog) induced only 2 antagonists of the NF-κB activityκB activity

• None of the preparations led to infiltration of immune cells in the duodenal biopsies (immune tolerance).

Probiotics, inflammatory bowel disease and irritable Probiotics, inflammatory bowel disease and irritable bowel syndrome

(McFarland and Dublin 2008, Heilpern D and Szilagyi A 2008, Moayyedi P el al. 2008 )

• VSL#3* has been effective in controlled clinical trials in preventing the onset of acute pouchitis in patients with newly formed surgical pouches and in maintaining remission following antibiotic treatment of acute pouchitis in antibiotic treatment of acute pouchitis in patients with a history of refractory or recurrent pouchitis

• Probiotics decrease abdominal symptoms in adults with irritable bowel syndrome (NNT 4)A h t t i i mix f 4 diff t • A yoghurt containing a mix of 4 different probiotic strains (Lactobacillus rhamnosus GG, L. rhamnosus Lc705, Propionibacterium freudenreichii ssp. shermanii JS ja Bifidobacterium animalis ssp. lactis Bb12) d s d bd min l p in nd dist nsi n nd decreased abdominal pain and distension and stabilized gut microbiota in patients with irritable bowel syndrome (Kajander et al. 2008).

* a mixture of four strains of lactobacilli: Lactobacillus casei, L. plantarum, L. acidophilus and L. delbrueckii subsp. bulgaricus, three strains of bifidobacteria: Bifidobacterium longum, B. breve and B. infantis and Streptococcus salivarius subsp. Figure from www.uoaa.org

Probiotics in the prevention of necrotizing t litis (NEC)entrocolitis (NEC)

(Deshpande G et al. 2007)

• Randomized double-blind placebo-controlled Randomized, double blind placebo controlled clinical trial with a probiotic mixture (Bifidobacterium infantis, Bifidobacteria bifidum, Bifid b t i l L t b ill id hilBifidobacteria longum, Lactobacillus acidophilus,each 2.5 x 109 CFU) twice a day

• The mixture was used from the beginning of The mixture was used from the beginning of enteral feedings till discharge (n=186)

The probiotic mixture had beneficial effects on pfeeding tolerance, duration of hospital stay,

frequencies of death, NEC and sepsis

Table from J Trop Pediatr 2008 Oct 8 [Epub ahead of print]Table from J Trop Pediatr. 2008 Oct 8. [Epub ahead of print]

Gut microbiota differences precede allergic f manifestations (Kalliomäki 2009, in press)

Reference

Study population

Definion of allergy Fecal analysis

Results

Kalliom�ki 2001

76 newborns with family history of allergy - 22 became cases and 54 controls

One or more positive skin prick test at 12 months

Gas-liquid chromatography and bacterial culture at 3 weeks and 3 months, FISH at 3 weeks

Different bacterial fatty acid profiles at 3 weeks, no difference in colonization patterns Higher counts of clostridia and tendency to lower counts of bifidobacteria by FISH, lower ratio of bifidobacteria to clostridia by FISH

Bj�rksten 2001 44 newborns Atopic eczema and/or at least 1 Bacteriological culture at 1 week andLower prevalence of bifidobacteria (during the 1st year) and t - 18 became cases and 26 controls positiveskin prick test at3,6,12,or

24 months 1, 3, 6 and12months

enterococci (duringthe1st month), lower counts ofbacteroides(at 12 months), higher prevalence of S. aureus (at 6 months) and higher counts of clostridia (at 3 months)

Penders 2006

78 newborns (prospective nested case-control) - 26 became cases and 52 controls

Eczema (based on ISAAC-questionnaire) and specific IgE at least to 1 allergen at 12 months

PCR-DGGE and quantitative real-time PCR at 1 month

E. coli more prevalent in infants later developing atopic eczema, no difference in total bacterial profiles or in bifidobacterial counts or bifidobacterial species composition

Penders 2007

957 newborns - over 30% developed eczema and approximately 10% recurrent wheeze - over a quarter became sensitized (at least 1 antigen-specific IgE cocentration > 0.3IU/ml)

Eczema or atopic eczema or at least 1 positive antigen-specific IgE (>0.3 IU/ml) or recurrent (>3) wheeze during the first 2 years of life

Quantitative real-time PCR at 1 month

Higher counts and prevalence of E.coli in infants later developing eczema, higher prevalence of C. difficile in infants later developing eczema, recurrent wheeze and sensitization

g p g )

Adlerberth 2007

324 newborns - 23% developed atopic eczema - 26% became sensitized

Atopic eczema, total and food-specific IgE levels at 18 months

Rectal cultures at 3 days, stool cultures at 1, 2 and 4 weeks and at 2, 6 and 12 moths

Neither atopic eczema nor food-specific IgE by 18 months of age were associated with time of acquisition of any particular bacterial group

Wang 2008

35 newborns - 15 became cases and 20 remained healthy

Atopic eczema at 18 months Terminal restriction fragment length polymorphism(T-RFLP) andTTGE

A reduced diversity of early gut microbiota of infants with atopic eczema

y

p y p ( )analysis of amplified 16S rRNA genes at 1 week

p

Probiotics in the treatment and prevention of allergic diseaseDisease/Marker Author and year Participants Type of probiotic(s) Duration of Type of study Outcomey p yp p ( )

intervention yp y

Eczema/atopic eczema (AE) Majamaa et al. 1997 27 infants Lactobacillus rhamnosus GG (LGG) 1 month R, DB, PC Severity of AE decreased Eczema/atopic eczema Isolauri et al. 2000 27 infants LGG or B. bifidum Bb-12 2 months R, DB, PC Severity of AE decreased Eczema/atopic eczema Rosenfeldt et al. 2003 43 children L. rhamnosus 19070 and L. reuteri DSM 6 weeks R, DB, PC Extent but not severity of AE decreased Eczema/atopic eczema Viljanen et al. 2005 230 infants LGG or a mixture of 4 probiotics 4 weeks R, DB, PC Severity of AE decreased only in IgE-

associated eczema in LGG group

Eczema/atopic eczema Weston et al. 2005 53 infants L. fermentum VRI-033 PCC 8 weeks R, DB, PC Severity of AE decreased Eczema/atopic eczema Sistek et al. 2006 59 infants L. rhamnosusand B. lactis 18 weeks R, DB,PC Severity of AE decreased only in childrenp y y

sensitised to food Eczema/atopic eczema Brouder et al. 2006 50 infants LGG or L. rhamnosus 3 months R, DB, PC No effect Eczema/atopic eczema F�lster-Holst et al. 2006 54 infants LGG 2 months R, DB, PC No effect Eczema/atopic eczema Gr�ber et al. 2007 102 infants LGG 12 weeks R, DB, PC No effect Asthma Wheeler et al. 1997 15 adults L. acidophilus 1 month DB, CO No effect on clinical parameters Allergic rhinitis (AR) Helin et al. 2002 36 adults LGG 5.5 months R, DB, PC No effect Allergic rhinitis Wang et al. 2004 80 adults L. paracasei-33 1 month R, DB, PC Quality of life improved Allergic rhinitis Peng et al 2005 90 adults L paracasei 33 1 month R DB PC Quality of life improvedAllergic rhinitis Peng et al. 2005 90 adults L. paracasei-33 1 month R, DB,PC Quality of life improvedAllergic rhinitis Ishida et al. 2005 49 adults L. acidophilus L-92 8 weeks R, DB, PC Decrease in nasal symptoms Allergic rhinitis Xiao et al. 2006 44 adults B. longum BB 536 13 weeks R, DB, PC Decrease in nasal symptoms Allergic rhinitis Tamura et al. 2007 109 adults L. casei strain Shirota 10 weeks R, DB, PC No effect Asthma and allergic rhinitis Giovannini et al. 2007 187 children L. casei 12 months R, DB, PC Decrease in rhinitis episodes Serum ECP, Ig E, eosinopenia Moreira et al. 2007 141 adults LGG 4 months R, DB, PC No effect Inflammatory markers in AR Ivory et al. 2008 10 adults L. casei strain Shirota 5 months R, DB, PC Decrease in antigen-specific IgE and

antigen-induced cytokines

Prevention of AE Kalliom�ki et al 2001 132 infants LGG 7 months R DB PC Incidence of eczemadecreased during thePrevention of AE Kalliom�ki et al. 2001 132 infants LGG 7 months perinatally

R, DB,PC Incidence of eczemadecreased during the first 2 years of life

Prevention of AE Kalliom�ki et al. 2003 107 infants same cohort same cohort same cohort Incidence of eczema decreased during the first 4 years of life

Prevention of AE Kalliom�ki et al. 2007 116 infants same cohort same cohort same cohort Incidence of eczema decreased during the first 7 years of life

Prevention of allergic disease Kukkonen et al. 2007 Kuitunen et al. 2009 (in press)

925 infants 4 probiotic strains and galactoolico-saccharide (a prebiotic)

7 months perinatally

R, DB, PC Incidence of eczema and AE decreased during the first 2 years of life. No effect at 5 years (except decrease in AE in cesarean-d li d hild )

delivered children)Prevention of AE Taylor et al. 2007 188 infants L. acidophilus (LAVRI-A1) 6 months

postnatally R, DB, PC No effect on incidence of eczema, but

increased allergic sensitisation

Prevention of allergic disease Abrahamsson et al. 2007 188 infants Lactobacillus reuteri 13 months perinatally

R, DB, PC Less AE during the second year of life

Prevention of AE Kopp et al. 2008 94 infants LGG 7 months perinatally

R, DB, PC No effect on AE or sensitisation, but increased risk for recurrent wheezy bronchitis

Prevention of AE Wickens et al. 2008 474 infants L. rhamnosus HN001 or 7 months R, DB, PC Lactobacillus decreased risk of AE,

Abbreviations: CO, cross-over; DB, double-blind; PC, placebo-controlled; R, randomized

B. animalis subs. Lactis strain HN019 perinatally whereas Bifidobacterium had no effect by 2years. No effect on sensitisation

Prevention of AE Soh et al. 2008 (in press) 253 infants B. longum (BL999) and L. rhamnosus

6 months R, DB, PC No effect on eczema or sensitisation during the first year of life

Probiotics in the treatment of eczema/atopic eczema

Disease Reference n Probiotic(s) Duration Type of study

Outcome

Eczema/atopic eczema (AE)

Majamaa et al. 1997

27 infants

Lactobacillus rhamnosus GG (LGG)

1 month R, DB, PC

Severity of AE decreased

Eczema/atopic Isolauri et al. 2000 27 f

LGG or B. bifidum Bb-12 2 months R, DB, C

Severity of AE decreased eczema

infants PC

Eczema/atopic eczema

Rosenfeldt et al. 2003

43 children

L. rhamnosus 19070 and L. reuteri DSM

6 weeks R, DB, PC

Extent but not severity of AE decreased

Eczema/atopic eczema

Viljanen et al. 2005 230 infants

LGG or a mixture of 4 probiotics

4 weeks R, DB, PC

Severity of AE decreased only in IgE-associated

eczema in LGG group

Eczema/atopic eczema

Weston et al. 2005 53 infants

L. fermentum VRI-033 PCC 8 weeks R, DB, PC

Severity of AE decreased

Eczema/atopic eczema

Sistek et al. 2006 59 infants

L. rhamnosus and B. lactis 18 weeks R, DB, PC

Severity of AE decreased only in children sensitised to

food

Eczema/atopic eczema

Brouder et al. 2006 50 infants

LGG or L. rhamnosus 3 months R, DB, PC

No effect

Eczema/atopic eczema

F�lster-Holst et al. 2006

54 infants

LGG 2 months R, DB, PC

No effect

Abbreviations: DB, double-blind; PC, placebo-controlled; R, randomized

Eczema/atopic eczema

Gr�ber et al. 2007 102 infants

LGG 12 weeks R, DB, PC

No effect

Probiotics in the treatment of allergic rhinitis/asthma

Disease Reference n Probiotics Duration Type of study

Outcome

Asthma

Wheeler et al. 1997

15 adults L. acidophilus 1 month DB, CO No effect on clinical parameters

Allergic rhinitis (AR)

Helin et al. 2002

36 adults LGG 5.5 months

R, DB, PC

No effect

Allergic rhinitis

Wang et al. 2004

80 adults L. paracasei-33 1 month R, DB, PC

Quality of life improved

Allergic rhinitis Peng et al 2005 90 adults L paracasei 33 1 month R DB Quality of life improvedAllergic rhinitis

Peng et al. 2005

90 adults L. paracasei-33 1 month R, DB, PC

Quality of life improved

Allergic rhinitis

Ishida et al. 2005

49 adults L. acidophilus L-92

8 weeks R, DB, PC

Decrease in nasal symptoms

Allergic rhinitis Xiao et al. 2006 44 adults B. longum BB 536

13 weeks R, DB, PC

Decrease in nasal t

536 PC

symptoms

Allergic rhinitis

Tamura et al. 2007

109 adults L. casei strain Shirota

10 weeks R, DB, PC

No effect

Asthma and allergic rhinitis

Giovannini et al. 2007

187 children L. casei 12 months R, DB, PC

Decrease in rhinitis episodes

Serum ECP, Ig E, eosinopenia

Moreira et al. 2007

141 adults LGG 4 months R, DB, PC

No effect

Inflammatory markers in AR

Ivory et al. 2008 10 adults L. casei strain Shirota

5 months R, DB, PC

Decrease in antigen-specific IgE and antigen-induced cytokines

Abbreviations: CO, cross-over; DB, double-blind; PC, placebo-controlled; R, randomized

Probiotics in the prevention of allergic disease

Disease Reference n Probiotics Duration Type of study

Outcome

Prevention of AE Kalliomäki et al. 2001

132 infants

LGG 7 months perinatally

R, DB, PC Incidence of eczema decreased during the first 2,

Kalliomäki et al. 2003

107 i f t

4, and 2003 infants

Kalliomäki et al. 2007

116 infants

7 years of life

Prevention of allergic disease

Kukkonen et al. 2007 Kuitunen et al. 2009

925 infants 895 infants

4 probiotic strains and galactoolicosaccharide (a prebiotic)

7 months perinatally

R, DB, PC Incidence of eczema and AE decreased during the first 2 years of life. No effect at 5 years (except decrease in AE in

2009 infants (except decrease in AE in cesarean-delivered children)

Prevention of AE Taylor et al. 2007 188 infants

L. acidophilus (LAVRI-A1) 6 months postnatally

R, DB, PC No effect on incidence of eczema, but increased allergic sensitisation

Prevention of allergic Abrahamsson et 188 Lactobacillus reuteri 13 months R, DB, PC Less AE during the second year gdisease al. 2007 infants perinatally

, , g yof life

Prevention of AE Kopp et al. 2008 94 infants LGG 7 months perinatally

R, DB, PC No effect on AE or sensitisation, but increased risk for recurrent wheezy bronchitis

Prevention of AE Wickens et al. 2008

474 i f t

L. rhamnosus HN001 or B i li b L ti

7 months i t ll

R, DB, PC Lactobacillus decreased risk of AE h Bifid b t i

Abbreviations: DB double blind; PC placebo controlled; R randomized

2008 infants B. animalis subs. Lactisstrain HN019

perinatally AE, whereas Bifidobacterium had no effect by 2 years. No effect on sensitisation

Prevention of AE Soh et al. 2008 (in press)

253 infants

B. longum (BL999) and L. rhamnosus

6 months R, DB, PC No effect on eczema or sensitisation during the first year of life

Abbreviations: DB, double-blind; PC, placebo-controlled; R, randomized

Conclusions• All probiotics are not equal but effects are strain-specific• Certain probiotic strains shorten the duration of viral diarrhea in

children by approximately one day• Probiotics have several properties that may assist in the development

of immature mucosal immunity and gut microbiota after birthof immature mucosal immunity and gut microbiota after birth• Prevention and treatment of allergic diseases with probiotics is an

interesting and active field of research• Preventive and therapheutic effects of certain probiotic strains on Preventive and therapheutic effects of certain probiotic strains on

pouchitis have been promising• Prevention of NEC in prematures and treatment of irritable bowel

syndrome are potential future indications for probioticsy p p• Well-designed and -conducted clinical studies are needed to expand

rationalized use of probiotics